Effects of combinations of curcumin, linalool, rutin, safranal, and thymoquinone on glucose/serum deprivation-induced cell death.

OBJECTIVE
Several phytochemical agents have been known to exhibit a neuroprotective effect. Among them, curcumin, linalool, rutin, safranal, and thymoquinone were widely investigated and neuroprotective activity of each of them was shown by several studies. This work was planned to investigate whether different combinations of them could induce better neuroprotective effect against glucose/serum deprivation (GSD)-induced cytotoxicity.


MATERIALS AND METHODS
PC12 cells were cultivated for 8 h in GSD condition in both the absence and presence of curcumin, linalool, rutin, safranal, thymoquinone, or combinations of them. At the end of the experiment, the cell viability was determined using MTT assay.


RESULTS
The cells cultured in GSD condition showed a significant decrease in viability (28±1%) as compared with those cultured in standard condition (100±2%). In the presence of curcumin (10 µg/ml), linalool (16 µg/ml), rutin (200 µg/ml), safranal (50 µg/ml), and thymoquinone (1 µg/ml), the cell viability increased to 69±3.4% (p<0.001), 44±1.4% (p<0.01), 64±0.5% (p<0.001), 49±2% (p<0.001), and 70±3.2% (p<0.001), respectively. When different combinations of the agents were tested, the best cytoprotective activity was obtained from safranal + curcumin + thymoquinone (97±5%, p<0.01 vs. untreated cells).


CONCLUSIONS
The present study demonstrated that a combination of safranal + curcumin + thymoquinone can block GSD-induced cell death and has the potential to be considered for management of cerebral ischemia and neurodegenerative diseases.


Introduction
Deprivation of cultured neurons from glucose and serum is a reliable in vitro model for development of new products for management of cerebral ischemia and neurodegenerative disorders (Ghorbani et al., 2011;Mousavi et al., 2010a;Sadeghnia et al., 2012). Cerebral ischemia is caused by restriction of blood flow to the brain, resulting in deficient supply of oxygen, glucose, and serum, thus leading to neuronal damage (Broughton et al., 2009). Currently, limited treatments exist for the management of neuronal damage following cerebral ischemia. Therefore, the search for new therapeutics is continued. Phytochemicals have always been good candidates to find new therapeutic drugs. Several phytochemical agents have been known to protect neurons against ischemic insult. Among them, curcumin, linalool, rutin, safranal, and thymoquinone are widely investigated and neuroprotective effects of each of them were shown by several studies. Curcumin (diferuloylmethane) is the major active constituent of Curcuma longa, a spice employed as a coloring and flavoring supplement in many foods. Evidence suggests that curcumin has antioxidant, antiinflammatory, neuromodulatory, and cytoprotective effects (Mendonca et al., 2012;Park et al., 2008;Rui et al., 2008;Tamaddonfard, 2012;Tamaddonfard et al., 2012).
Linalool, a plant-derived monoterpene alcohol, is a component of a number of essential oils including lavender, coriander, and sweet basil. The beneficial effect of linalool on nervous disorders is well documented (Batista et al., 2010;Devi et al., 2007;Elisabetsky et al., 1995). Rutin, a flavonoid constituent of foods and plantbased beverages, has several pharmacological properties including cytoprotective (Janbaz et al., 2002), anticonvulsant (Nassiri-Asl et al., 2008, and strong antioxidant (La Casa et al., 2000) activities. Furthermore, neuroprotective effect of rutin has been shown against neuronal death induced by focal ischemia and by ischemia-reperfusion injury (Khan et al., 2009;Pu et al., 2007).
Safranal, a monoterpene aldehyde, is the main constituent of the essential volatile oil in Crocus sativus and responsible for the characteristic aroma and odor of this plant (Tarantilis et al., 1995). Recently, it has been demonstrated that safranal and C. sativus have neuroprotective effects in vitro and in vivo studies (Fukui et al., 2011;Hosseinzadeh et al., 2005;Hosseinzadeh and Sadeghnia, 2007;Mousavi et al., 2010b;Mousavi and Bathaie, 2011).
Thymoquinone is the natural main constituent of the volatile oil in Nigella sativa seeds. The protective effect of N. sativa and thymoquinone against glucose/serum deprivation (GSD)-induced neuronal damage has also been reported by Mousavi and colleague (2010a). Based on the evidence mentioned above, we hypothesized that combinations of curcumin, linalool, rutin, safranal, and thymoquinone may have higher neuroprotective effect and can be used as therapeutic agent for cerebral ischemia and neurodegenerative diseases. Therefore, the present work was carried out to investigate whether different combinations of these phytochemicals are capable to protect neuronal cells against GSD-induced cell death.

Cell viability assay
The cell viability was determined using MTT assay as previously described (Hadjzadeh et al., 2006;Mortazavian and Ghorbani, 2012;Rakhshandeh et al., 2012). Briefly, aliquots of 10 µl of MTT solution (5 mg/ml) were added to culture medium and the reaction mixture incubated for 2 h. The mixture was removed and the resulting formazan was dissolved by adding 100 µl dimethyl sulfoxide to each well of the plate. The optical density of formazan dye was read at 570 and 620 nm (background) using a StatFAX303 plate reader. The percentage of viable cells was calculated as a ratio of optical density of treated to control (cultured in standard medium only) cells.

Statistical analysis
All data were expressed as mean±SEM. One way ANOVA followed by Tukey's post hoc test for multiple comparisons were used for statistical evaluation. Statistical significance was accepted at p<0.05.

Discussion
In this study, glucose and serum limitation-induced damage in PC12 cells were used to partially model the pathological process of cerebral ischemia in an attempt to search a phytochemical compound with potent neuroprotective effect. Our results showed that all the tested agents (curcumin, linalool, rutin, safranal, and thymoquinone) are able to inhibit neurotoxic effect of GSD condition. These results are in agreement with previous studies demonstrated neuroprotective effects of curcumin (Mendonca et al., 2012;Park et al., 2008;Rui et al., 2008), linalool (Batista et al., 2010;Devi et al., 2007;Elisabetsky et al., 1995), rutin (Khan et al., 2009;Pu et al., 2007), safranal (Fukui et al., 2011;Hosseinzadeh et al., 2005;Hosseinzadeh et al., 2007;Mousavi et al., 2010b), and thymoquinone (Mousavi et al., 2010a). Effects of curcumin and rutin were seen in a concentration-dependent manner. However, the best cytoprotective activities of safranal and thymoquinone were observed at low concentrations. It may be due to the cytotoxicity of high concentrations of safranal and thymoquinone (Abdullaev et al., 2003;Behravan et al., 2010;Khader et al., 2009).
We hypothesized that combinations of curcumin, linalool, rutin, safranal, and thymoquinone may have better neuroprotective effect. To test this thesis, all possible combinations of these agents, 26 mixtures, were exposed to the cells cultured in GSD condition. Most of the combinations could decrease the GSD-induced cell death. However, only linalool+curcumin, thymoquinone+curcumin, rutin+curcumin+thymoquinone, safranal+curcumin+thymoquinone, and curcumin+linalool+rutin+safranal+thymoqui none compounds were able to induce better neuroprotective effect than curcumin or thymoquinone which showed the best effect in monotherapy. Curcumin is a common constituent of these compounds and its combination with thymoquinone and safranal could restore the cell viability to the normal level. On the other hands, some mixtures (linalool+thymoquinone, rutin+thymoquinone, safranal+thymoquinone, linalool+rutin+thymoquinone, linalool+safranal+thymoquinone, curcumin+linalool+safranal, rutin+safranal+thymoquinone, and linalool+rutin+safranal+thymoquinone) not only failed to enhance cell viability, but also abolished the cytoprotective activities of each individual agent. Although it is difficult to draw a conclusion from such complex finds, this effect may be due to possible interaction between thymoquinone and other agents when curcumin is absent.
In conclusion, our study revealed that curcumin and thymoquinone have the best neuroprotective effects among the tested phytochemicals; a combination of safranal+curcumin+thymoquinone can block GSD-induced cell death. Therefore, confirming these results in in vivo studies, this combination has the potential to be used as a new therapeutics for cerebral ischemia and neurodegenerative diseases.